1. Field of the Invention
This invention concerns basement-membrane proteins useful in adhering keratinocytes to the dermis. More specifically, this invention concerns a method of using these proteins to enhance the success of skin transplantation.
2. General Background of the Invention
The use of cultured epidermal grafts (keratinocyte grafts) to treat patients with life-threatening burns was first reported by O'Conner et al., The Lancet 1:75-78 (1981). Small skin biopsy specimens from burn patients were cultured in vitro, and the cultured autografts were placed on full thickness wounds on the arms of burn patients. The cultured keratinocytes successfully grew to cover the wounds in six weeks. Subsequent attempts have been made to improve this method by modifying it to grow keratinocytes in serum-free medium. Others have suggested using composite cadaver skin allografts resurfaced with autologous cultured keratinocytes. Attempts have also been made to use different backing materials for the cultured cells or to vary the keratinocyte culture methodology. The results of cultured keratinocyte transplants, however, have often been disappointing.
One of the most useful applications for keratinocyte grafts has been in patients with burns damaging more than half of the body surface. Such patients have insufficient donor sites to provide enough split skin thickness grafts to resurface the area of the burn after surgical excision. Unfortunately, the results of keratinocyte autografting in these circumstances have been variable and disappointing. Cultured epidermal grafts have been found to be significantly more fragile than normal skin and more prone to blistering. Woodley et al., JAMA 259:2566-2571 (1988). Some researchers have suggested that an abnormality in one or more connective tissue components within the autografts might explain the altered epidermal-dermal adherence observed clinically. The identity of that component, however, has remained obscure.
Laminin is a previously described noncollagenous glycoprotein. This molecule is a high molecular weight (850 kDa) extracellular matrix glycoprotein found almost exclusively in basement membranes. The basement membrane is a ubiquitous, specialized type of extracellular matrix that separates organ parenchymal cells from interstitial collagenous stroma. Interaction of cells with this matrix is an important aspect of both normal and neoplastic cellular processes.
Laminin purified from the murine Engelbreth-Holm-Swarm (EHS) tumor, is a disulfide bonded trimer consisting of a 400 kDa A chain, a 220 kDa B1 chain and a 210 kDa B2 chain (Cooper et al., Eur. J. Biochem. 119:189-197 (1981)). By rotary shadowing electron microscopy, EHS laminin has the image of an asymmetric cross with one long arm and three short arms (Engel et al., J. Mol. Biol. 150:97-120 (1981)). Fragmentation studies of the large EHS laminin molecule have facilitated the localization of several of its properties to individual molecular domains. The large size and multidomain structure of this molecule give it the potential to span the basement membrane, mediate the interactions of multiple basement membrane components, and interact with receptors at basal cell surfaces adjacent to basement membrane. Several extracellular matrix proteins are capable of interacting with EHS laminin, including type IV collagen, nidogen, and heparin sulfate proteoglycan.
Many types of cells including keratinocytes (Stanley et al., J. Invest. Dermatol. 82:456-459 (1982)) and dermal fibroblasts (Woodley et al., J. Cell. Physiol. 136:140-146 (1988)) have been shown to synthesize laminin in culture. Some cell lines, including choriocarcinoma cells (Peters et al., J. Biol. Chem. 260:14732-14742 (1985)) and HT 1080 fibrosarcoma cells (Alitalo et al., Cell 19:1053-1062 (1980)) synthesize an excess of B chains relative to A chain. Pertinent to these observations, recent in situ hybridization experiments of human skin samples have revealed abundant expression of B1 and B2 chain genes, but undetectable expression of A chain gene (Olsen et al., Lab Invest. 60:772-782 (1989)). It is possible that in both skin and cultured cells, B1 and B2 chains are synthesized in relative excess and that synthesis of A chains serves as the rate limiting step for laminin assembly.
Additionally, laminin chains are apparently assembled into a variety of structures. Merosin is a laminin variant which contains a B1 chain, a B2 chain, and a third chain distinct from the A chain, although it shares 40% homology by sequence analysis (Ehrig et al., Proc. Natl. Acad. Sci. 87:3264-3268 (1990)). Mouse heart laminin is a laminin variant with a substituted A chain of a size similar to the one in merosin (Paulsson and Saladin, J. Biol. Chem. 264:18726-18732 (1989)). S-laminin, another laminin variant, contains a normal A chain, B2 chain, and a variant chain that shows some sequence homology to the B1 chain (Hunter et al., J. Cell Biol. 113:971-978 (1989)). Recently, the merosin variant chain and the S-laminin variant chain have been found complexed together with B2 in certain tissues, including the myotendonous junction (Engvall et al., J. Cell Biol. 1:731-740 (1990)). Two other laminin variants which apparently lack an A chain have been reported, but unlike merosin and S-laminin, it is not known whether they are present in tissue. These include rat RN22 schwannoma laminin (Davis et al., J. Neurosci. 5:2662-2671 (1985); Edgar et al., J. Cell Biol. 106:1299-1306 (1988); and 3T3 adipocyte laminin, Aratani and Kitigawa, J. Biol. Chem. 263:16163-16169 (1988)). These forms contain B1 and B2 subunits, but lack electrophoretically normal A subunits. Thus laminin exists as a family of proteins. Its individual members have restricted tissue distributions, for example merosin and S-laminin localize to muscular and neural basement membranes respectively, but not to epithelial basement membranes.
Laminin influences the growth and differentiation of many types of cells, and is present at the earliest stages of human development. Laminin is also a component of the extracellular matrix deposited by keratinocytes onto culture substratum (Carter et al., J. Cell Biol. 111:3141-3154 (1990); Marchisio et al., J. Cell Biol. 112:761-773 (1991)). Exogenously supplied EHS laminin facilitates the attachment of a variety of epithelial cell types (Terranova et al., Cell 22:719-726 (1980); Goodman et al., J. Cell Biol. 105:595-610 (1987)), including human keratinocytes (Wilke and Skubitz, J. Invest. Dermatol. 97:141-146 (1991)) but markedly decreases motility of cultured keratinocytes (Woodley et al., J. Cell. Physiol. 136:140-146 (1988)). This marked reduction in the motility of cultured keratinocytes is an impediment to the use of EHS laminin as an adhesion protein for transplanted keratinocytes. Inhibition of keratinocyte migration would diminish the ability of a cultured sheet of keratinocytes to spread out over a wound surface and completely cover an epidermal defect. Hence EHS laminin is not believed to be suitable for use in keratinocyte transplantation.
It is an object of this invention to identify and provide a therapeutically useful form of a newly isolated connective tissue component that provides epidermal-dermal adherence.
It is another object of this invention to use such a therapeutically useful substance to enhance the adhesion of transplanted cultured keratinocytes to an underlying substrate, such as a mammalian or human dermis.
Yet another object is to provide such a therapeutic substance that has minimal inhibition of keratinocyte migration.
These and other objects of the invention will be understood more clearly by reference to the following detailed description.